High-Yield Synthesis and Purification of an -Helical Transmembrane Domain Lillian E. Fisher* ,1 and Donald M. Engelman† ,2 *Department of Chemistry and †Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520 Received December 22, 2000; published online May 1, 2001 Polypeptides corresponding to hydrophobic trans- membrane -helices, such as residues 69 –101 of glycoph- orin A, are notoriously difficult to prepare in quantities sufficient for biophysical experiments. Simple synthetic and purification approaches reported here have been developed by combining a few modifications to standard procedures, without resorting to elevated temperatures, expensive activation strategies, or complex hydrophobic solvent mixtures. The cost of screening projects, prepar- ing labeled peptides, and examining sequence varia- tions is thereby significantly reduced. The quality of the peptide synthesized by this small-scale 9-fluorenylme- thoxycarbonyl (Fmoc) strategy is comparable to that of the peptide synthesized by an experienced resource fa- cility using a large-scale tert-butyloxycarbonyl strategy. Using reverse-phase HPLC, the desired peptide was sep- arated from the primary side product (a Leu or Ile dele- tion) and quantitatively recovered at greater than 98% purity. Baseline resolution was achieved using a water: acetonitrile gradient to elute the peptides from a cyano- propyl column at ambient temperature. Combining these approaches readily yields 10 to 20 mg of pure transmembrane peptide from a small-scale Fmoc syn- thesis. The approaches are readily transferable to trans- membrane sequences not previously synthesized and do not require setting up a specialized facility. The time and start-up expense required to launch new studies are thereby reduced expanding the range and detail with which questions in membrane protein biophysics can be explored. © 2001 Academic Press Key Words: hydrophobic peptides; -helix; solid-phase peptide synthesis; SPPS; Fmoc; reverse-phase HPLC. Examination of genome sequences for stretches of predominately hydrophobic amino acids 15–23 resi- dues in length suggests that as much as 30% of open reading frames encode proteins that may cross mem- branes using hydrophobic helices (1, 2). Despite the abundance of membrane proteins and the important roles they play in many cellular processes, investiga- tion of membrane protein folding and assembly lags far behind studies of soluble protein folding. Use of the tools of molecular biology to express predicted trans- membrane domains, either directly or as chimeric fu- sion proteins (3), has a number of potential limitations. The gene products may be toxic to the host, may form insoluble aggregates, or may be rapidly degraded thereby preventing accumulation of the polypeptide. Thus, facile chemical synthesis emerges as a desirable approach. Development of reliable synthetic approaches uti- lizing a tert-butyl (Tboc) 3 protection strategy has provided a source of crude material. The cost of a Tboc synthesis, however, limits the number of trans- membrane peptide sequence variations that can be studied. In addition, the necessary use of hydro- fluoric acid to cleave the peptides from the resin limits the approach to specialized facilities. Although a small-scale 9-fluorenylmethoxycarbonyl (Fmoc) strategy can reduce the cost and eliminate the use of hydrofluoric acid, synthesis of transmembrane pep- tides using an Fmoc strategy is not yet routine (4 – 6). Common features of natural transmembrane se- quences still present significant synthetic chal- 1 Current address: Memorial Sloan-Kettering Cancer Center, Box 251, RRL 513, 1275 York Avenue, New York, NY 10021. 2 To whom correspondence should be addressed at Department of Molecular Biophysics & Biochemistry, Yale University, P.O. Box 208114, New Haven, CT 06520-8114. Fax: (203) 432-6381. E-mail: don@paradigm.csb.yale.edu. 3 Abbreviations used: DIPEA, N,N-diisopropylethylamine; Fmoc, 9-fluorenylmethoxycarbonyl; GpA, glycophorin A; HBTU, O-benza- triazol-1-yl-N,N,N',N'-tetramethyluroniumhexafluorophosphate; HOBT, hydroxybenzatriazole; HPLC, high-pressure liquid chroma- tography; MALDI-TOF, matrix-assisted laser desorption time of flight mass spectrometry; PEG-PS, polyethyleneglycol–polystyrene; Tboc, tert-butyloxycarbonyl; TFA, trifluoroacetic acid. 102 0003-2697/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved. Analytical Biochemistry 293, 102–108 (2001) doi:10.1006/abio.2001.5122, available online at http://www.idealibrary.com on